The development of automated counters for counting red blood cells, white blood cells and platelets, together with the increased demand for quality control in the clinical laboratory, produced a need for stabilized cellular components of blood to assess the reproducibility and accuracy of counters. A large number of compounds have been employed to increase the rigidity of the cell membrane of these cellular components so that the cells do not lyse on aging. Examples of these agents: formaldehyde, tannic acid, glutaraldehyde, and pyruvic aldehyde have been used for this purpose.
Platelet reference controls are commercially available from both human and equine platelets. The platelets are removed from the blood by centrifugation, washed with buffered saline and then "fixed" with glutaraldehyde. The final platelet reference control product has a shelf life of at least 6 months at room temperature. The platelet reference control is used in the following manner:
1. It is mixed well to insure uniformity.
2. A micro-pipet is used to remove 5 ul (microliter) of platelets. The 5 ul is placed in 15.0 ml of an isotonic diluent. The exact formula of the diluent differs from company to company, but usually contains sodium chloride, potassium chloride, and a phosphate buffer to maintain the pH at 7.3.
3. The container for the dilution is usually made of polystyrene-disposable plastic with a polyethylene snap-on lid. After adding the
PLATELETS TO THE DILUENT THE CONTAINER IS GENTLY INVERTED 2-3 TIMES AND THE PLATELET MIXTURE IS COUNTED. The platelet count under these circumstances is very reproducible (.+-.2%).
However, if the container is agitated vigorously or is allowed to stand for 30 minutes, a decrease in count is observed. For example, counts of 8400-8900 are achieved by gentle inversion while counts of 5600-6700 result from vigorous shaking. Even just setting for 30 minutes will reduce a count from 11,200-11,500 to 10,000-10,100.
The foregoing data is typical of data obtained from samples allowed to set or samples that are vigorously shaken, however, considerable variation of the decrease in platelet count occurs particularly in the samples that are shaken because of the difficulty of calibrating the manner of shaking.
It was postulated that the decrease in platelet counts on shaking or standing was due to adsorption of the platelets on the polystyrene surface. Therefore, glass and silicon coated glass were tested in the same manner as the polystyrene containers and were also found to decrease the platelet counts with time and upon shaking.
Next, an alternative was considered consisting of treatment of the plastic containers with organic solvents such as chloroform, acetone or methylene chloride. This treatment has to be a quick rinse since the solvents, if left in contact with the styrene container, would dissolve it. However, these treatments increased the adsorption of the platelets, viz., a decrease in count.
The increased adsorption following treatment with the solvent suggested that the organic solvent was removing the mold release agent, which is found on the surface of the polystyrene container. Release agents are sprayed on the mold at intervals to assist in obtaining release of the plastic container from the mold. Varying amounts are present on the plastic containers. To pursue this idea further, several mold release agents were sprayed on the interior of the polystyrene-plastic containers used for counting the platelets; the containers were then filled with diluent and platelets and then shook. It was felt that if the release agent were responsible for the variation in counts found from one container to another and for the more rapid decrease in counts after treatment with organic solvents, then, following spraying of the release agent on the container, there should be no decrease in platelet count. It was found that a container once rinsed with trichloro-methane and dried thereafter did not achieve the desired stability, the count dropping from 9900 after gentle inversion to 6300 when shaken vigorously 6 times. However, a mold release agent MS-122 consisting of a fluorocarbon telomer available from Miller-Stephenson Chemical Co., Inc. of Danbury, Conn. produced 10,239 counts after gentle inversion and 10,182 counts after 6 vigorous shakes.
After discussion with several manufacturers of mold release agent, I finally concluded that it might be the surfactant properties of the mold release agents that prevented the adsorption of platelets to the polystyrene surface. For this reason, I examined a variety of cationic, anionic and nonionic surfactants. The cationic surfactants invariably caused aggregation of the platelets. Most of the nonionic and anionic surfactants prevented the decrease in counts that occur on standing or agitation of the platelets. Although the solution to our problem appeared to be at hand, an examination of the size of the surfactant treated platelets in a Coulter ZBI, an instrument which is used for size analysis of particles such as platelets and white blood cells, indicated that there was a large decrease in the size of the platelet. However, when the platelets were examined under the microscope with an ocular-micrometer, no change in size of the platelet could be determined. The principle of the Coulter Counter is that it measures changes in conductivity; the surfactants alter the conductivity and thus make the platelet appear smaller to the instrument. All of the surfactants tested up to this point produced this apparent change in size. The decrease in apparent size is an unacceptable change since most instruments used to count platelets would count them inaccurately under these circumstances.
I thereupon tried a series of polyethylene glycols of which there are several. There is polyethylene glycol 400, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 20,000. The number following polyethylene glycol is the approximate molecular weight of the polyethylene glycol. Polyethylene glycols are polymers of ethylene oxide with the generalized formula HOCH.sub.2 (CH.sub.2 OCH.sub.2)n CH.sub.2 OH, n represents the average number of oxyethylene groups. All of the polyethylene glycols decrease surface tension. The liquid polyethylene glycols (200, 300, 400) when added to platelet suspensions did not prevent adsorption of the platelets, that is, a decrease in count still occurs. The PEG-4000, 6000 and 20,000 all were effective in preventing the decrease in counts upon shaking. This was puzzling since the effect on surface tension is similar for all PEG products. Both liquid and solid PEGs in aqueous solutions have surface tensions of about 70 dynes per centimeter at 25.degree. C. at low concentrations and drop to 45-50 dynes/cm at 30-70%.
By the use of a minor amount of solid polyethylene glycol (0.1-0.5 grams) per liter of the platelet reference control dilution, it is possible to stabilize the control against count decrease on either standing or vigorous agitation and without apparent decrease in size which would result in a false low count in instrument counters.
Following the discovery that the minor amounts of polyethylene glycol would stabilize the platelet reference control a search of the art was undertaken to determine how polyethylene glycols had been used previously in conjunction with blood generally and platelets in particular. The search revealed the use of surface active substances such as "Pluronics". These are poly (oxypropylene) poly (oxyethylene) condensates with molecular weights in the range 1,000-15,000 and are produced by BASF Wyandotte. The use of these was reported in Bibliothica Anatomica, No. 12, pages 208-212 (1973). Although the addition of the Pluronics to the platelet reference controls prevented aggregation or freezing and adsorption to the container, however, within 10 minutes after addition of the Pluronic to the platelets, the apparent shape started to decrease. It was surprising since the close relationship of Pluronics and polyethylene glycol suggested that the Pluronics might also be satisfactory.
Another prior art reference uncovered in the search also had to do with Cryopreservation Techniques, Transfusion May-June, 1975, Volume 15, No. 3, pages 219-225. This also had to do with the use of additives to platelets for freezing purposes and indicated that dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG) would be useful with the DMSO being superior. However, the DMSO failed to stabilize the platelets.